There is a fundamental gap in understanding the mechanisms of gene-environment interactions in the pathogenesis of craniofacial defects. The existence of this gap hinders our ability to prevent defects in at risk individuals. Our long-term goal is to understand the underlying mechanisms that predispose patients to craniofacial defects in order to design targeted preventive interventions. A mouse model deficient in Twisted gastrulation (TWSG1) allows us to study the underlying mechanisms of these interactions because Twsg1-/- mice manifest HPE with a low penetrance of about 17%, which increases to 100% after exposure to subteratogenic doses of the retinoic acid (RA). The objective of this study is to characterize the mechanisms of increased susceptibility of TWSG1-deficient mice to RA-induced HPE. The central hypothesis is that dysregulation of bone morphogenetic protein (BMP) signaling in Twsg1 mutants reduces an apoptotic threshold, sensitizing the embryos to teratogenic effects of RA. Disrupting apoptotic pathways would be expected to be embryo-protective. The rationale for using this model is that environmental factors, unlike genetic aberrations, are modifiable in humans as long as the underlying mechanisms can be identified. Based on our preliminary data, the central hypothesis will be tested by pursuing three specific aims: 1) to examine the effects of in utero RA exposure on the molecular program that governs neural and midline facial development in Twsg1 mutant embryos; 2) to examine the impact of RA exposure on BMP signaling and cellular processes underlying the HPE phenotype in Twsg1 mutant embryos; 3) to understand the role of oxidative stress and p53 activation in mediating BMP/RA effects and their potential as targets for HPE prevention. The proposed research presents a novel concept that the BMP pathway may regulate embryonic susceptibility to environmental insults, such as RA. In addition, this study begins to address the role of p53 signaling in development, which is currently poorly understood. Finally, the preventive interventions that this project seeks to develop may have an impact on the current clinical practice. The expected outcomes of this study are: 1) elucidation of the mechanisms of gene-environment interactions in the pathogenesis of HPE, specifically how mutations with low penetrance can sensitize the embryos to environmental teratogens; 2) improved understanding of the interactions between BMP and RA signaling; 3) enhanced knowledge about the roles of the oxidative stress and p53 activation in HPE; and 4) establishment of a model for testing preventive interventions for embryonic teratogens. Such results are expected to have an important positive impact on prevention of craniofacial defects.